Regulatory CD4 T cells (Treg) play a key role in the prevention of autoimmunity and maintenance of allograft tolerance. For example, in the gut, Treg prevent effector T cells from mounting a destructive inflammatory response to a myriad of non-pathogenic bacterial antigens. Natural Treg arise in the thymus and exhibit a CD45RBLo phenotype characteristic of activated cells. Treg can also be induced, for example, by activation of peripheral T cells in vitro in the presence of Antigen Presenting Cells plus cytokines or in vivo by oral antigen. However, it is generally unclear whether these Treg primarily arise as an outgrowth of preexisted Treg within the CD45RBLo population vs. de novo generation from naive CD45RBHi effector cells. Recent findings suggest that the latter is possible, at least in specialized circumstances. If Treg can be induced de novo, we can delineate the signals involved in differentiation from effector cells to Treg. This would have important clinical implications since this conversion represents a shift in the immune response from immunity to tolerance. Moreover, CTLA-4 upregulation is accompanied by induction of Foxp3, a transcription factor key for Treg development. These changes occur in vitro in isolated T cells and in the absence of T cell activation. Preliminary data reveal that this mAb can prevent IBD caused by CD45RBHI effectors by inducing Treg function, suggesting that alpha-CD45RB initiates de novo conversion of effector cells into Treg in the absence of T cell activation. In Project 2 we aim to determine the mechanism(s) of action of these novel Treg and delineate signals leading to Foxp3 and CTLA-4 induction. This is central to the theme of this PPG, which aims to define peripheral mechanism of tolerance. In Aim 1, we will define the mechanisms by which de novo Treg prevent IBD. In addition to secretion of suppressive cytokines we hypothesize that they inhibit both APCs and T effector cells through CTLA-4/B7 interactions. We will compare the role of CTLA-4/B7 and Foxp3 expression by both induced and natural Treg. Since only a subpopulation of alpha-CD45RB -treated cells expresses CTLA-4, in Aim 2 we will define surface markers that accompany cytoplasmic CTLA-4 expression that can further identify regulatory cells within the population. In Aim 3, we will define the signaling pathways that lead to Treg induction. These studies will provide novel insight into the immunobiology of Treg and the pathways leading to their induction. In turn, this will provide new targets for tolerance induction and the prevention of autoimmunity and transplant rejection. Project 2 integrates with other projects in this PPG on multiple levels.